CN103901143A - Pretreatment method for analyzing tetrabromobisphenol A in small amount of biologic serum - Google Patents
Pretreatment method for analyzing tetrabromobisphenol A in small amount of biologic serum Download PDFInfo
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- CN103901143A CN103901143A CN201410171617.XA CN201410171617A CN103901143A CN 103901143 A CN103901143 A CN 103901143A CN 201410171617 A CN201410171617 A CN 201410171617A CN 103901143 A CN103901143 A CN 103901143A
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Abstract
The invention discloses a pretreatment method for analyzing tetrabromobisphenol A in a small amount of biologic serum, relates to a detection and analysis technique of organic pollutant in a biologic sample, and provides a pretreatment technique for testing the content of tetrabromobisphenol A in serum. The pretreatment method comprises the following steps: adding the sample into methyl tertiary butyl ether and a n-hexane reagent which are mixed in an isovolumetric manner, washing by using a potassium chloride solution, drying in anhydrous sodium sulfate, concentrating the filtrate to be constant in weight, measuring the weight of fat, redissolving the sample, and subsequently removing fat impurities by using a Hybrid solid phase extraction column with zirconia granules. The detection on the sample by using an ultra-high performance liquid chromatography-tandem mass spectrometer shows that the method needs a relatively small amount of serum, is good in purification effect, excellent in fat removing effect, simple to operate and relatively high in recycling rate. By adopting the method, the tetrabromobisphenol A in a small amount of biologic serum sample can be effectively extracted and purified, and a basic method is provided for tetrabromobisphenol A environment and health research.
Description
Technical field
The present invention relates to the detection analytical technology of organic contaminant in biological sample, particularly relate to sample extraction, the purification method of analyzing tetrabromobisphenol A content in a kind of a small amount of biological anteserum.
Background technology
In recent years, tetrabromobisphenol A is widely used in household electrical appliance and articles for daily use as one of huge bromide fire retardant of use amount, as the electronic products such as computing machine, printer, mobile phone, televisor, washing machine and textile, foam furniture, building materials etc.According to European bromine science and environment forum (Brominated Science and Environmental Forum, BSEF) report, except Jordan, Israel, the U.S., Japan etc., Ye Shi tetrabromobisphenol A major country of production of China.Due to the production-scale continuous growth of Related product, containing the continuous increase of tetrabromobisphenol A electron wastes, and can be discharged into the increase of tetrabromobisphenol A quantity in environment, make people start to pay close attention to the potential problem of environmental pollution of tetrabromobisphenol A and the impact on health.
Investigation shows, because tetrabromobisphenol A uses extensively, all detect that in the world the tax of tetrabromobisphenol A deposits, and content is higher in the multiple surrounding medium such as soil, sediment, water body and the atmosphere of a lot of countries and regions and biological sample.Owing to frequently detecting tetrabromobisphenol A in surrounding medium and Biomedia, the public is extraordinarily paid close attention to the bio-toxicity of tetrabromobisphenol A.At present research finds that tetrabromobisphenol A and metabolic product thereof have potential bioconcentration in the situation that biosome exposes repeatedly, and the bio-toxicity such as cell, immunity and endocrine interference.In recent years, tetrabromobisphenol A is considered to the potential incretion interferent of a kind of worth discussion and concern, also has research to think that tetrabromobisphenol A has latency environment persistence and bioaccumulation, may cause persistency organic contaminant problem.The Europe protection the Atlantic, northeast marine environment the convention (OSPAR) is not cancelled so far after calendar year 2001 is included tetrabromobisphenol A in optimal control pollutant, in its report, thinks that tetrabromobisphenol A has persistence pollution problem.Based on this, the research of carrying out tetrabromobisphenol A environment and health aspect is significant, particularly study the concentration of tetrabromobisphenol A in biosome serum and by zoopery simulate tetrabromobisphenol A enter biosome after the regularity of distribution in serum the impact research of tetrabromobisphenol A is had to vital role.
Because tetrabromobisphenol A concentration in biological anteserum sample is lower, and serum sample matrix more complicated, biological anteserum sample is can collection capacity less simultaneously, therefore the requirement of blood serum sample pretreatment technology is just become to stricter.A lot of samples before analysis not through well extracting and purified treatment, can not accurate response biological anteserum when analysis in tetrabromobisphenol A concentration, or due to the matrix effect considerable influence recovery.Therefore pretreatment technology is the key of tetrabromobisphenol A content technology in analytic sample, and directly affects the research and development of tetrabromobisphenol A health effectiveness.
Summary of the invention
The object of the invention is for the problem of biological anteserum sample pre-treatments complexity provide that a kind of amount of samples is few, the recovery is high, good impurity removing effect, extraction and cleanup technology rapidly and accurately, weaken matrix interference effect, realize the quantitative test to tetrabromobisphenol A in a small amount of biological anteserum sample.Difference with the prior art of the present invention is, adopt the mode of cell ultrasonication to extract tetrabromobisphenol A in blood serum sample, the solid phase extraction column that employing contains zirconia particles is removed lipid impurity in serum, has ensured extraction efficiency and has avoided complicated purifying step to lose tetrabromobisphenol A content in sample.
To achieve these goals, technical scheme of the present invention is as described below.
A kind of pre-treating method (Fig. 1) that is applicable to analyze tetrabromobisphenol A in a small amount of biological anteserum sample, comprises the following steps:
(1) accurately pipette serum, add tetrabromobisphenol A, watery hydrochloric acid and the isopropyl alcohol of C13 mark;
(2) methyl tert-butyl ether and the normal hexane reagent that add equal-volume to mix, through the ultrasonic extraction of cell Ultrasonic Cell Disruptor;
(3) put the interior low-speed centrifugal of hydro-extractor 5 minutes, shift out upper strata extract;
(4) twice of repeating step (2)-(3);
(5) merge No. three extracts, be transferred to separating funnel;
(6) add Klorvess Liquid fully to shake, after leaving standstill, collect respectively organic phase and water, in triplicate;
(7) merge the water of collecting for three times, the methyl tert-butyl ether and the normal hexane reagent that add equal-volume to mix, fully, after concussion, collect organic phase;
(8) organic phase of generation in collection step (6)-(7), after anhydrous sodium sulfate drying, concentrated to Rotary Evaporators, transfers to the glass centrifuge tube of known weight, and nitrogen dries up to constant weight;
(9) weigh containing the glass tube of sample, use gravimetric determination fat weight;
(10) weigh after sample methyl alcohol redissolve, Hybrid solid phase extraction column through containing zirconia particles is removed lipid impurity, collect trickle, Nitrogen evaporator is concentrated into after certain volume, filter with 0.2 μ m filtrator, after filtration, detect tetrabromobisphenol A concentration to Ultra Performance Liquid Chromatography-tandem mass spectrum combined instrument.
Described in the method, biology comprises animal used as test, wild animal and crowd.
In this method step (2), cell Ultrasonic Cell Disruptor ultrasound intensity is 25%, ultrasonic 4 seconds, stop 3 seconds, and after totally 7 minutes, remove extract, in triplicate.
Advantage of the present invention and good effect are: sample is after the ultrasonic extraction of cell Ultrasonic Cell Disruptor, use potassium chloride washing extract, for ensureing that the recovery adopts organic solvent back extraction cleansing solution, after washing, extract is concentrated into the dry fat weight that weighs after anhydrous sodium sulfate drying, then after redissolving with methyl alcohol, the Hybrid solid phase extraction column through containing zirconia particles is removed fat.In blood serum sample, the removal of lipid impurity is a step important in pre-treatment program, if can not effectively eliminate, can affect testing result and pollute instrument.The required serum amount of this method is less, good purification, grease removal effect excellence, simple to operate, the recovery is higher, adopts Ultra Performance Liquid Chromatography-tandem mass spectrometer to measure, detect and be limited to 0.01ng/g fat, be specially adapted to the mensuration of tetrabromobisphenol A content in a small amount of biological anteserum sample.
The recovery of standard addition of this pre-treating method is as shown in table 1.
The recovery of standard addition of this pre-treating method of table 1
Brief description of the drawings
Fig. 1 is analytical approach process flow diagram;
Fig. 2 is canonical plotting;
Fig. 3 is the recovery comparison diagram of the different extracting modes of blood serum sample;
Fig. 4 is the different recovery comparison diagrams that purify except lipid impurity mode of blood serum sample.
Embodiment
Below in conjunction with embodiment, technical scheme of the present invention is further described.
(1) pre-treatment of blood serum sample
Accurately pipette serum, add tetrabromobisphenol A, 2mL6moL/L hydrochloric acid and the 6mL isopropyl alcohol of 10ng C13 mark, the methyl tert-butyl ether and the normal hexane reagent that add 20mL equal-volume to mix, through the ultrasonic extraction of cell Ultrasonic Cell Disruptor, cell Ultrasonic Cell Disruptor ultrasound intensity is 25%, ultrasonic 4 seconds, stop 3 seconds, totally 7 minutes, put afterwards in hydro-extractor with the speed of 3000 rpms centrifugal 5 minutes, shift out upper strata extract, in triplicate; Merge No. three times extract, be transferred to separating funnel, adding 20mL mass concentration is 1% potassium chloride, and solution fully shakes washing, collects respectively organic phase and water, in triplicate after leaving standstill; Methyl tert-butyl ether and normal hexane reagent that the aqueous portion of collecting adds 20mL equal-volume to mix fully shake back extraction, after leaving standstill, collect organic phase; Merge all organic phase anhydrous sodium sulfate dryings, after Rotary Evaporators is concentrated, transfer to the glass centrifuge tube of known weight, nitrogen dries up to constant weight; Weigh containing the glass tube of sample, use gravimetric determination fat weight; After weighing, sample redissolves with methyl alcohol, Hybrid solid phase extraction column (Supelco company of the U.S.) through containing zirconia particles is removed lipid impurity, collect trickle, after Nitrogen evaporator is concentrated into 1mL, filter with 0.2 μ m PTFE filtrator (Pull company of the U.S.), after filtration, detect tetrabromobisphenol A concentration to Ultra Performance Liquid Chromatography-tandem mass spectrum combined instrument.
(2) utilize Ultra Performance Liquid Chromatography-Tandem Mass Spectrometry Analysis tetrabromobisphenol A concentration
Instrument model: Ultra Performance Liquid Chromatography-tandem mass spectrum combined instrument ACQUITY UPLC-MS/MS (Waters company of the U.S.), comprises quaternary gradient liquid chromatography pump, high pressure binary gradient pump, and Masslynx4.1 workstation.
Chromatographic condition: the ACQUITY UPLC BEH C18 chromatographic column (1.7 μ m, 500mm × 2.1mm I.D) that adopts Waters company of the U.S. to produce; 40 DEG C of column temperatures; Flow velocity 0.30mL/min; Sample size 10 μ L; Mobile phase A is ultrapure water, and Mobile phase B is methyl alcohol; Adopt gradient elution, initial concentration is 70%B, and 0～0.5min is that 70%B linearity is elevated to 100%B, and 0.5～2min is 100%B, and 2min is down to 70%B moment, and 2～3min is 70%B, and the overall operation time amounts to 3min.
Mass spectrum condition: adopt Negative electrospray ionization detecting pattern (ESI-); Capillary voltage is 3.5kV; Ion source temperature is 120 DEG C; Desolventizing gas is nitrogen (N
2); Taper hole blowback air flow velocity is 50L/h; Desolventizing temperature degree is 350 DEG C; Desolventizing gas velocity is 800L/h; Collision cell pressure is 3.8 × 10
-3mbar; Mass analyzer low side resolution LM1 is that 13.0, LM2 is 13.0; High-end resolution HM1 is 13.0V, and HM2 is 13.0V; Ion energy 1 is 1.0,2 to be 4.0; Adopt MRM multi-channel detection.
(3) drafting of typical curve
Get the blank blood serum sample mixing, respectively sample is processed according to the pre-treating method of blood serum sample described in step (1).After pending end, configure the tetrabromobisphenol A typical curve of serial matrix tetrabromobisphenol A and C13 mark with the blank blood serum sample obtaining.The peak area ratio of the tetrabromobisphenol A with tetrabromobisphenol A to C13 mark and concentration value do quantitative criterion curve (Fig. 2), in order to the amount of analyte in calculation sample.
(4) mensuration of sample concentration and the recovery
Blood serum sample is collected in the rat that experimental article is Wistar.18 rats are raised one week in experimental animal room endoadaptation, observed without extremely rear and be divided at random 3 groups, be respectively control group, low dose group, high dose group.Rat does not process in check plot, low dose group rat per os gavage at 8 o'clock in morning every day gives the sodium carboxymethyl cellulose suspension of the tetrabromobisphenol A of 10mg/kg dosage, high dose group rat per os gavage at 8 o'clock in morning every day gives the sodium carboxymethyl cellulose suspension of the tetrabromobisphenol A of 30mg/kg dosage, after successive administration 90 days, by rat anesthesia, heart puncturing extracting blood.Desired blood room temperature leave standstill 15 minutes, 3000 rpms centrifugal 10 minutes, separate upper serum, be placed in insulation can and take back rapidly laboratory, in-80 DEG C of low temperature refrigerators, preserve.According to step (1), sample is carried out to ultrasonic extraction afterwards, extract washs through potassium chloride, and with organic solvent back extraction cleansing solution, after washing, extract is concentrated into the dry fat weight that weighs after anhydrous sodium sulfate drying, then removes lipid impurity through the Hybrid solid phase extraction column of chromatogram section of the U.S. after redissolving with methyl alcohol.Detect through Ultra Performance Liquid Chromatography-tandem mass spectrometer afterwards, contrast the content that obtains tetrabromobisphenol A in serum with step (3) Plays curve.In peak area ratio substitution step (3) the Plays curve of the tetrabromobisphenol A of the tetrabromobisphenol A obtaining with sample detection to C13 mark, the concentration of trying to achieve is the concentration of tetrabromobisphenol A in sample.
Meanwhile, in the peak area of the tetrabromobisphenol A of middle C13 mark and same concentrations typical curve, the peak area of the tetrabromobisphenol A of C13 mark contrasts per sample, calculate recovery rate.The method recovery is calculated according to the following formula:
R＝(A/A
0)×100％
The R-recovery, %;
The peak area of the tetrabromobisphenol A of C13 mark in A-working sample;
The tetrabromobisphenol A peak area of C13 mark under same concentrations in A0-typical curve.
Utilize the present invention to detect per os and expose tetrabromobisphenol A concentration in various dose tetrabromobisphenol A rat blood serum, result is as shown in table 2.
Tetrabromobisphenol A concentration in the actual blood serum sample of table 2
| Dosage group | Sample size n | Tetrabromobisphenol A concentration (μ g/g, fat) |
| Check plot | 6 | 0.00±0.00 |
| Low dose group | 6 | 19.53±6.63 |
| High dose group | 6 | 38.11±22.56 |
Claims (3)
1. a pre-treating method of analyzing for a small amount of biological anteserum tetrabromobisphenol A, is characterized in that: the method comprises the steps,
(1) accurately pipette serum, add tetrabromobisphenol A, watery hydrochloric acid and the isopropyl alcohol of C13 mark;
(2) methyl tert-butyl ether and the normal hexane reagent that add equal-volume to mix, through the ultrasonic extraction of cell Ultrasonic Cell Disruptor;
(3) put the interior low-speed centrifugal of hydro-extractor 5 minutes, shift out upper strata extract;
(4) twice of repeating step (2)-(3);
(5) merge No. three extracts, be transferred to separating funnel;
(6) add Klorvess Liquid fully to shake, after leaving standstill, collect respectively organic phase and water, in triplicate;
(7) merge the water of collecting for three times, the methyl tert-butyl ether and the normal hexane reagent that add equal-volume to mix, fully, after concussion, collect organic phase;
(8) organic phase of generation in collection step (6)-(7), after anhydrous sodium sulfate drying, concentrated to Rotary Evaporators, transfers to the glass centrifuge tube of known weight, and nitrogen dries up to constant weight;
(9) weigh containing the glass tube of sample, use gravimetric determination fat weight;
(10) weigh after sample methyl alcohol redissolve, Hybrid solid phase extraction column through containing zirconia particles is removed lipid impurity, collect trickle, Nitrogen evaporator is concentrated into after certain volume, filter with 0.2 μ m filtrator, after filtration, detect tetrabromobisphenol A concentration to Ultra Performance Liquid Chromatography-tandem mass spectrum combined instrument.
2. the pre-treating method as described in right 1 requirement, is characterized in that: described in the method, biology comprises animal used as test, wild animal and crowd.
3. the pre-treating method as described in right 1 requirement, is characterized in that: in step (2), cell Ultrasonic Cell Disruptor ultrasound intensity is 25%, ultrasonic 4 seconds, stop 3 seconds, and after totally 7 minutes, shift out extract, in triplicate.
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| CN108572222A (en) * | 2017-03-13 | 2018-09-25 | 中国科学院生态环境研究中心 | Method for detecting the organic brominated flame-retardant of the trace in sample |
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| CN108572222A (en) * | 2017-03-13 | 2018-09-25 | 中国科学院生态环境研究中心 | Method for detecting the organic brominated flame-retardant of the trace in sample |
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